Epac (exchange protein directly activated by cAMP): a drug target and biomarker in chronic lymphocytic leukemia

Fiona Murray, Anja Zahno, Lingzhi Zhang, Alexandra Anastasopulos, Laura Rassenti, Thomas Kipps, Paul Insel

University of California, San Diego, La Jolla, California, USA

Chronic lymphocytic leukemia (CLL) is characterized by deficient apoptosis of B-cells. Patients with CLL show differences in their clinical course, which is classified as ”indolent” or “aggressive”. Adenosine 3’5’ cyclic-monophosphate (cAMP) promotes apoptosis of peripheral blood mononuclear cells (PBMC) isolated from patients with CLL (Tiwari et al., 2004). The actions of cAMP are largely mediated by protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). PKA has been proposed to be pro-apoptotic, while Epac to increase the survival of malignant cells. We hypothesized that the differential expression of Epac and Epac-dependent signalling in CLL alters the apoptotic effect of cAMP and allow it to be a novel target or aid in classification of disease prognosis.

Blood was collected from healthy donors and CLL patients following informed consent, in agreement with institutional guidelines, and PBMC and B-cells were isolated as described (Endo et al., 2007). Epac-1 and Epac-2 mRNA and protein expression were assayed in these cells and in EHEB cells, a CLL-cell line, using real-time PCR and immunoblotting. Epac activity was assayed by Rap-1 activation after incubation with an Epac-selective cAMP analogue [8-pCPT-2Me-cAMP (8Me), 50μM, 15min, Rap-1 activation assay]. [3H]thymidine incorporation and FACS (annexin 5 staining) were used to assess DNA synthesis and apoptosis, respectively, in cells treated with 8Me (50μM, 48h) or the PKA selective cAMP analogue, N6- Phenyladenosine-cAMP (N6, 50μM, 48 h). Data are expressed as mean ± SEM. Statistical comparisons were performed using paired/unpaired Student’s t-tests, with P<0.05 considered significant.

QPCR revealed that Epac-1 is more abundant than Epac-2 in normal, CLL and EHEB cells. Compared to normal PBMC, CLL cells (645 +/- 184-fold n=34, P<0.01), but not purified B-cells (6.7 +/- 5-fold, n=7, NS), have increased Epac1 mRNA expression. Epac-1 mRNA expression was increased in both indolent and aggressive CLL (398.1 +/- 105.5, n=7; 700.5 +/- 121.0, n=27, fold-increase vs. normal P<0.01 respectively). Epac-1 protein expression was increased in CLL cells vs. normal PBMC (˜14-fold increase vs. PBMC, n=16, P<0.01). 8Me stimulated Rap-1 activation but was not pro-apoptotic in CLL cells while PKA-specific cAMP analogues killed CLL cells. . In EHEB cells activation of Epac and PKA had opposite effects: Epac activation increased proliferation (60% ± 5.7 increase vs. vehicle, n=3, P<0.05), whereas PKA activation decreased proliferation (50.7% ± 2.9 decrease vs. vehicle, n=3 P<0.05).

We conclude that CLL cells (from both indolent and aggressive patients) have increased Epac-1 mRNA and protein expression compared to normal PBMC. Epac activation, unlike PKA activation, does not induce apoptosis of CLL cells and increases proliferation of EHEB. These data suggest that decreasing the expression and function of Epac-1 may be beneficial for the treatment of CLL by increasing the pro-apoptotic effects of drugs that act through raising cAMP and that activate PKA

Endo T. et al., (2007) Blood. 109(2): 703-10

Tiwari S. et al., (2004), Blood. 103(7): 2661-7